Determining sequential micellization steps of bile salts with multi-CMC modeling.

Area of Science:

• Biochemistry
• Physical Chemistry
• Chemical Physics

Background:

• Bile salts form complex aggregates in aqueous solutions.
• Previous studies focused on a single critical micelle concentration (CMC), leaving the stepwise nature of bile salt aggregation unresolved.
• Understanding the discrete aggregation steps and CMC values is crucial for comprehending bile salt behavior.

Purpose of the Study:

• To investigate the concentration-dependent micellization of bile salts.
• To resolve multiple, sequential critical micelle concentrations (CMCs) in bile salt solutions.
• To develop and apply a novel multi-CMC model for analyzing bile salt aggregation.

Main Methods:

• Utilized Nuclear Magnetic Resonance (NMR) chemical shift titrations.
• Developed and applied a multi-CMC phase separation modeling approach.
• Correlated phase separation and mass action models to analyze micellization steps.

Main Results:

• Resolved multiple closely spaced CMCs (preliminary, primary, secondary) in dihydroxy and trihydroxy bile salt systems.
• Identified four distinct CMCs for deoxycholate below 100 mM at pH 12 (3.8 ± 0.5 mM, 9.1 ± 0.3 mM, 27 ± 2 mM, 57 ± 4 mM).
• Observed three CMCs in other bile salt systems under basic conditions, with the model explaining complex NMR data and revealing chemical shifts of dark states.

Conclusions:

• Bile salt aggregation proceeds through discrete, sequential steps rather than a continuous process.
• The developed multi-CMC model accurately resolves multiple CMCs and characterizes distinct micellar states.
• This work provides a more comprehensive understanding of bile salt micellization, resolving long-standing questions about aggregation.